The cystic fibrosis conductance regulator (CFTR) is a polytopic integral membrane protein that is a member of the ABC transporter supergene family. These proteins are composed of two cytosolic nucleotide binding domains (NBDs) and two transmembrane domains (TMDs) that fold and associate to form the functional ABC enzymes which utilize ATP binding and hydrolysis to mediate transmembrane events. In contrast to other ABC transporters, CFTR also contains a unique, additional, largely-disordered portion called the R region. Mutations in the gene that encodes CFTR cause cystic fibrosis, typically by disrupting the folding and assembly of the protein into its functional native structure. The misfolded mutant protein is recognized by quality control proteins in the cell that target it for degradation. The most common of these mutations is ?F508 which is found on the surface of NBD1 in a region that mediates interactions with the TMDs in other ABC transporters. Thus, three fundamental questions about the molecular pathology of the ?F508 mutation arise. Does the mutation disrupt the folding of the NBD1? Does the mutation disrupt the association of NBD1 and other domains of CFTR or its partner proteins? How is CFTR that is not efficiently folded identified and what is the committed recognition step? Three specific aims are proposed to address these questions and, further, to provide insight into the more general process of polytopic membrane protein folding. They are: Define the effects of the ?F508 mutation on the folding and dynamics of NBD1. The native states of wild type and ?F508 NBD1 are remarkably similar, but the mutant more readily populates a partially folded state. The structure and dynamics of this state will be characterized. Other residues that interact with F508 to affect conversions between these states will be identified using computational and genetic approaches. Elucidate the effects of the ?F508 mutation on the interactions of NBD1 with other domains of CFTR. Recent crystal structures indicate that the 508 side chain is exposed on the surface of NBD1 near a predicted interface with intracellular loops (ICLs) connecting the transmembrane spans of CFTR. Using cell biological, biochemical, structural, and computational methods, the ICLs that interact with NBD1 will be identified and the effect of the ?F508 mutation on the strength of the interaction determined. Extant methods for monitoring interactions with the R region and other domains will also be utilized. Identify the proteins that interact with the nascent CFTR chain at the earliest steps of folding and those that interact when folding is disrupted by disease-causing mutations such as ?F508. A powerful biosynthetic method of incorporating site-specific photoaffinity probes in defined CFTR nascent chains will be used to identify proteins that interact during the earliest steps of folding and integration. Of particular interest are the identities of the proteins preliminary data indicate preferentially recognize nascent chains with specific CF-causing mutations. These studies are necessary for a fundamental understanding of the development of membrane protein structure and strategies to circumvent the molecular pathology of CF. PUBLIC HEALTH RELEVANCE. Most cases of cystic fibrosis, a common fatal genetic disease, are caused by mutations that interfere with the assembly of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The studies proposed will elucidate the details of how the disease-causing mutations interfere with this process. Understanding the assembly process, and, thus, the detailed molecular pathology, will provide important information for developing targeted therapeutics for cystic fibrosis.